Comparative analysis of antigen coding genes in 15 red cell blood group systems of Yunnan Yi nationality in China: A cross‐sectional study

Abstract Introduction There are few analyses of the 15 red blood group system antigen coding genes found in the Yunnan Yi nationality. This has caused many poteintial dangers relating to clinical blood transfusion. In this report, the coding genes and distribution of 15 blood group antigens system in the Yi nationality were tested and compared with those of Han nationality and other ethnic minorities. Methods The samples came from the healthy subjects in the first people's Hospital of Qujing, Yunnan Province. Two hundred and three Yunnan Yi and 197 Han nationality individuals were included. Thirty‐three blood group antigens with a low frequency from the 15 blood group systems of Yunnan Yi blood donors were genotyped and analyzed by PCR‐SSP. Sanger sequencing was used to detect A4GALT from the Yunnan Yi nationality. The χ 2 test was used to analyze observed and expected values of gene distribution to verify conformation to the Hardy‐Weinberg equilibrium law. Fisher's exact test was used to analyze gene frequency distribution, and the statistical significance was set at p < 0.05. Results The ABO blood group examination results for the Yi nationality and the local Han nationality in Qujing City, Yunnan Province, showed the majority were type A and type O, while the least prevalent was type AB. RhD+ accounts for more than 98% of the Yi and Han populations. There was a significant difference in ABO blood group antigen distribution between these two nationalities (p < 0.05), but there was no significant difference in the composition ratio of D antigen in the Rh blood group system (p > 0.05). Compared with Tibetan (Tibet), Zhuang (Nanning), and Dong (Guangxi), the gene distribution frequencies of Rh blood group system phenotype CC were significantly lower in the Yunnan Yi nationality (p < 0.05). There were significant differences in six erythrocyte phenotypic antigens in the Yi nationality in Yunnan compared with Han nationality, such as LW(a−b−), JK(a−b+), MMSs, Di(a−b+), Wr(a−b−), and Kp(a−b+) (p < 0.05). There were gene phenotypes with a low frequency in the four rare blood group systems: LW, MNS, Wright, and Colton. Several different mutation types occurred in the P1PK blood group system's A4GALT gene. Conclusion Yunnan Yi nationality has a unique genetic background. There are some significantly different distributions of blood group system genes with a low frequency in different regions and groups in China. Multiple mutations in the A4GALT gene of the P1PK blood group system may be related to their environment and ethnic evolution.

Several different mutation types occurred in the P1PK blood group system's A4GALT gene.
Conclusion: Yunnan Yi nationality has a unique genetic background. There are some significantly different distributions of blood group system genes with a low frequency in different regions and groups in China. Multiple mutations in the A4GALT gene of the P1PK blood group system may be related to their environment and ethnic evolution.

| INTRODUCTION
The red cell blood group systems is of great importance to clinical transfusion medicine, and until now 43 have been identified by International Transfusion Association (ISBT). 1 The genetic background of the erythroid blood group system is polymorphism, and the gene frequency distribution is related to ethnicity and region. 2 Erythroblood group system antibodies cause fetal and neonatal hemolytic disease (hemolytic disease of the fetus and newborn, HDFN) and hemolytic transfusion response (hemolytic transfusion reactions, HTRs).
The ABO blood group system is one of the most important human erythroid blood group systems. 3 It consists of four antigens (A, B, A1 and A, B). These antigens, called oligosaccharide antigens, are widely expressed in erythrocyte membranes, tissue cell membranes, and in saliva and humoral fluids. 4 It is important for the diagnosis and treatment of cross-mating, neonatal hemolysis, and organ transplantation. The Yi of Yunnan province have a unique genetic background, the antigen distribution of its blood group system with a low frequency has not until now been fully reported, and there is no large sample of gene polymorphism in Yunnan Yi population. We found two cases of P phenotype in a previous study, and gene sequencing found a new A4GALT allele c.456_457_in-sACACCCC homozygous mutation (NCBI number: MG812384), which is the molecular formation mechanism of the p phenotype. 5 A4GALT polymorphisms and rare Au(a−b+) individuals were also found in this pedigree. Therefore, it is speculated that A4GALT and blood type antigen genes with a low frequency have multiple polymorphisms in the Yunnan Yi population.
It was reported that the antigen of the P1PK blood group system was not confirmed by the ISBT until 2011. The system has three antigens, respectively: P1, P k , and NOR. 6 The locus of the P1PK antigen is sub-band 2 in region 1 of the long arm of chromosome 22,22q. 7,8 This gene is called A4GALT. The gene product is: 4-galactosyltransferase, consisting of 353 amino acids. The A4GALT gene is polymorphic with 52 alleles, with most gene mutations occurring on exon 3. [9][10][11] The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal galactose α1-4Gal of P K (Gb3/CD77) glycosphingolipid α1-4Gal, which plays an important role in transfusion medicine, obstetrics and pathogen susceptibility. 12 Anti-P1 antibodies is associated with a hemolytic transfusion response, whereas P and PK-related antibodies are associated with hemolytic transfusion response, neonatal hemolytic disease, and spontaneous abortion. 13,14 In addition to the P1Pk blood group system, whether the distribution of other blood group systems is unique in the Yunnan Yi population deserves our study. For example, the Rh blood group system contains two genes, RhD and RhCE, and the expressed antigen is a 12-transmembrane glycoprotein. Due to RhD blood group incompatibility, neonatal hemolysis will occur. 15,16 The Yi nationality is the largest ethnic minority in Yunnan, China, with a unique genetic background due to ethnic migration and integration. In this study, PCR-SSP genotype analysis of P1PK blood type A4GALT, and the study of distribution characteristics in their gene polymorphisms, provide a data basis and assist in the establishment of a comprehensive rare blood type database.

| METERIALS AND METHODS
2.1 | Blood samples 2.3 | PCR amplification A PCR reaction system was prepared, and bidirectional primers were added at a concentration of 10 pmol/L (Supporting Information: Table S1 for the primer sequence). The PCR products were subjected to agarose electrophoresis, and the target fragments were cut in a 2.0 ml centrifuge tube and recovered using a glue recovery kit. The recovered products were sequenced.

| Genotyping detection
Detect 12 clinically important erythrocyte antigen genes by RT-qPCR with human erythrocyte rare blood group genotyping kit, add specific primers (Supporting Information: Table S1) to amplify PA and PB genes by PCR, then sequence amplified products by Sanger sequencing, and finally compare the sequencing results to obtain the mutation point (mutation position) and then determine the genotype.

| Detection of blood groups with a low frequency of Yunnan population in China
Rare blood type genotype and gene frequency in the Yunnan Yi nationality ( Table 1)

| Rare blood group phenotype distribution differences in the Yunnan Yi nationality andother ethnic minorities in different regions of China
There was significant difference between Yunnan Yi nationality and other ethnic minorities (p < 0.001). Compared with other ethnic minorities, the Yi nationality in Yunnan has significant difference (p < 0.05). (Table 3).

| DISCUSSION
In this study, results indicated that the Yi and local Han people in Qujing City, Yunnan Province are mostly A-type and O-type. There was a significant difference in the antigen distribution of ABO blood group system between the two ethnic groups (p < 0.05), while the D antigen composition ratio of Rh blood group system was not HE ET AL.   this may be related to the small number of individual samples collected in the clinic, resulting in certain deviations in the statistics. 21 The study of 15 erythrocyte rare blood group system antigen gene poly morphisms in Yunnan population not only provides date for human population genetics, ethnic migration and blood transfusion treatment of blood group patients, but also improves the construction of Yunnan rare blood group gene database, help to solve the problem of difficult blood type identification and cross-matching incompltibility, reduce blood transfusion reactions such as immune hemolysis, and provide a strong guarantee for clinical safe blood transfusion and precise blood transfusion therapy. 22 The results of this study suggest that in the LW blood group system, Lw(a−b+) and Lw(a+b+) blood groups were not detected in the Han population, while in the Yunnan Yi people, the Lw(a−b+) blood group accounted for 0.0099, the proportion of Lw(a+b+) blood group is 0.0099, which is statistically different from the distribution of the Han population in the LW blood group system. 23,24 In the 203 yunnan Yi people, the proportion of the MMss blood type population reached 0.3448, which was significantly higher than the proportion of the Han population of 0.2843. In terms of the NNss blood type, the proportion of the Yunnan Yi population was significantly reduced. In the MNS blood group system, it can be seen that the differences between different blood types are statistically significant. 25 In the Diego blood group system, it can be seen that the Di(a−b+) blood type is significantly increased in the Yunnan Yi population compared with the Han population, while the Di(a+b+) blood type is less in the Yunnan Yi population, with p < 0.05, which is statistically significant.
In the Wright blood group system, the expression of Wr(a+b−) and Wr(a+b+) blood groups were not detected in the Han population, while the frequencies of 0.0099 and 0.0493 were found in the Yunnan Yi population, respectively. The expression difference between the two populations has statistical significance. 26,27 In this study, the distribution of blood group system genotypes with a low frequency in Yunnan was found significantly different among ethnic minorities in different regions of China. 28 Subsequently, with the polymorphism of P antigen A4GALT gene polymorphism of P1PK was detected. P1PK blood group system antigen which has a common molecular basis, is encoded by the A4GALT gene. 29 Therefore, detecting A4GALT gene polymorphism is an effective method to understand the antigen distribution of P1PK in the Yi group. It has been reported that Yunnan Province is a region with a high incidence of thalassemia, and ethnic minorities have a higher carrying rate. 30 This means that the unique genetic background of ethnic minorities in Yunnan can easily lead to the occurrence of hematological diseases. In previous studies of two rare P phenotypes in Yi families, 5 it was found that they lack all antigens from the P1PK blood group system. Anti-P1PK (anti-Tja) antibody was present in all individuals with the P phenotype, which coagulates all phenotype red blood cells except the P phenotype, resulting in habitual abortion in women early in pregnancy. 31,32 The A4GALT gene determines the formation of the P phenotype, and its exon 3 variation which causes amino acid changes, may cause

CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare. All authors have read and approved the final version of the manuscript. They had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis. Kun-hua He affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; No important aspects of the study have been omitted; Any discrepancies from the study as planned have been explained.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ETHICS STATEMENT
This study was approved by the Medical Ethical Committee of Qujing No.1 Hospital of Yunnan Province, and the ethics number is IRB2018-001(S)-01.

TRANSPARENCY STATEMENT
The lead author Kun-Hua He affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.